Phagocytosis is a form of endocytosis wherein solid particles such as bacteria are engulfed by the cell membrane to form an internal phagosome. Phagocytosis is distinct from other forms of endocytosis, such as the vesicular internalization of liquids. Phagocytosis is a key mechanism used by the immune system to remove pathogens, cell debris, dead tissue cells and small mineral particles from circulation in the body.
Phagocytes are white blood cells that protect the body by phagocytosing harmful foreign particles, bacteria, and dead or dying cells, and thus are essential for fighting infections and developing subsequent immunity. Phagocytes of humans and other animals are called “professional” or “non-professional” depending on how effective they are at phagocytosis (Ernst & Stendahl, 2006, “Phagocytosis of Bacteria and Bacterial Pathogenicity”, Cambridge University Press: NY, p. 186). The distinguishing factor between professional and non-professional phagocytes is that professional phagocytes (such as neutrophils, monocytes, macrophages, dendritic cells, and mast cells) have surface receptors that can detect harmful objects, such as bacteria, that are not normally found in the body. The immune system recognizes invading cells (such as microbes and viruses) as foreign because these invading cells either express determinants that are absent on host cells or do not express “markers of self” that are normally present on host cells. Injected or implanted materials are also perceived as foreign as these invariably activate macrophages and other phagocytes, and this foreign body response occurs in spite of synthetic coatings such as those with polyethylene glycol (PEG) that are intended to maximize compatibility.
During an infection by a pathogen, chemical signals attract phagocytes to places where the pathogen has invaded the body. These chemical signals may come from bacteria or from other phagocytes already present. When phagocytes come into contact with bacteria, the receptors on the phagocyte's surface bind to them, leading to the engulfing of the bacteria by the phagocyte. Some phagocytes kill the ingested pathogen with oxidants and nitric oxide. After phagocytosis, macrophages and dendritic cells can also participate in antigen presentation, a process in which a phagocyte moves parts of the ingested material back to its surface. The antigen is then displayed to other cells of the immune system. Some phagocytes then travel to the body's lymph nodes and display the material to white blood cells called lymphocytes, a key event in the development of immunity.
Phagocytes are thus professional eating cells of the innate immune system and are responsible for protecting humans and other animals from attacks by foreign pathogens. However, phagocytes may also attack elements that have been intentionally introduced into the body, such as implants, artificial tissue, artificial organs and vesicles bearing therapeutic agents, and this may reduce their lifetime in the body. Furthermore, pathogens have evolved methods to evade attacks by phagocytes, such as covering themselves with molecules that deflect recognition and/or attachment of phagocytes.
A phagocyte may display many types of receptors on its surface, including opsonin receptors, scavenger receptors, and Toll-like receptors. Opsonin receptors increase the phagocytosis of bacteria that have been coated with immunoglobulin G (IgG) antibodies or with complement (a complex series of blood proteins that destroy cells or mark them for destruction). While scavenger receptors bind to a large range of molecules on the surface of bacterial cells, Toll-like receptors bind to more specific molecules, increasing phagocytosis and causing the phagocyte to release inflammatory hormones.
The protein signal regulatory protein-alpha (SIRP-alpha), also known as tyrosine-protein phosphatase non-receptor type substrate 1 or CD172A (cluster of differentiation 172A), is a member of the signal-regulatory-protein (SIRP) family and belongs to the immunoglobulin superfamily. SIRP family members are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of receptor tyrosine kinase-coupled signaling processes.
SIRP-alpha has been shown to be an inhibitory phagocyte receptor. Once it is activated, SIRP-alpha inhibits pro-phagocytic signals from Fc and complement receptors, resulting in inhibition of phagocytosis (de Almeida et al., 2009, Immunopharmacol. Immunotoxicol. 31(4):636-40).
Nanoparticles similar in size to viruses are frequently decorated with antibodies for targeted therapeutics or imaging. Although such particles are sufficiently small to avoid passive entrapment by capillaries in vivo, macrophages in the spleen and liver are well known to clear liposomes and nanoparticles (even when coated with PEG) within hours or days of injection into the circulation. This limits delivery to sites of disease. Whether phagocytosis of antibody-opsonized small particles (<500 nm) involves the same set of signals as larger particles remains a broad research question. A particle that activates SIRP-alpha could in principle limit phagocytic clearance in vivo as well as in vitro and also through help clarify lower size limits for its signaling in phagocytic pathways.
SIRP-alpha is highly polymorphic within both human and mouse (Strowing, et al., 2011, Proc. Natl. Acad. Sci. USA. 108(32):13218-23), with the likely consequence that this minimizes pathogen interactions with SIRP-alpha (Hatherley et al., 2008, Mol Cell 31(2):266-77). Among natural mouse variants, only NOD/SCID mice (and derived strains) express a mouse polymorph of SIRP-alpha that cross-reacts with human cells. This fact may explain why human blood cells engraft and circulate in these mice better than other strains of mice (Strowing, et al., 2011, Proc. Natl. Acad. Sci. USA. 108(32):13218-23; Legrand et al., 2011, Proc. Natl. Acad. Sci. USA. 108(32):13224-9). NSG mice thus provide an ideal platform for in vivo assessment of SIRP-alpha binding and activating ligands on synthetic particles.
There remains a need in the art to identify a novel method for controlling the activity of phagocytes and their interactions with foreign bodies. Such method should allow the labeling of foreign bodies with one or more molecules that would preclude or delay their recognition as foreign by phagocytes. This method would thus ensure that the labeled foreign body is not subjected to immediate phagocytosis and degradation once introduced in the body. The present invention fulfills these needs.